Citation or identification of any reference herein, or any section of this application shall not be construed as an admission that such reference is available as prior art to the present application. The disclosures of each of the publications cited herein, are hereby incorporated by reference in their entirety in this application, and shall be treated as if the entirety thereof forms a part of this application.
Worldwide, inflammatory and hyperproliferative diseases of the skin, gut, eye, vagina, mouth, nasopharyngeal region and bladder, including cancer, cause substantial morbidity and mortality. Conventional antinflammatory drugs such as corticosteroids offer one of the greatest means of preventing or treating inflammation. Unfortunately, many diseases remain without effective therapies. New therapies, including novel delivery methods and novel therapeutics are needed in order to meet the worldwide challenge of inflammation.
Inflammation is involved in a number of disease pathologies, including acne vulgaris, Alzheimer's, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis (RA), asthma, atherosclerosis, atopic dermatitis, atrophic vaginitis, autoimmune diseases, bacterial vaginitis, celiac disease, chronic prostatitis, cancer, colitis, Crohn's disease, dermatitis/eczema, diaper rash, diverticulitis, erythroderma, fibromyalgia, glomerulonephritis, hepatitis, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, irritable bowel syndrome (IBS), lichenoid disorders, lupus erythematous, nephritis, Parkinson's, pelvic inflammatory disease, psoriasis (including flexural, pustular, palmoplantar pustular, nail, acrodermatitis of hallopeau, psoriatic arthritis, and plaque psoriasis), reperfusion injury, rheumatoid arthritis, rosacea, sarcoidosis, sebaceous cysts, systemic lupus erythematous (SLE), transplant rejection, ulcerative colitis vasculitis, or chronic condition known as dystrophic epidermolysis bullosa (DEB), which causes severe blistering and can lead to early deaths from skin cancer.
Parasites and infectious agents are detriments to humans, animals including wild animals, pets and livestock, plants, food and/or the environment (soil, water, etc.) include protozoans, amoebas and helminthes, such as hookworm, intestinal nematodes (roundworms), tissue nematodes including onchocerciasis (river blindness), caused by the nematode Onchocerca volvulus, Trichinosis, Dracunculiasis, and the Filariases, Trematodes (Schistosomes and Other Flukes), Cestodes (Tapeworms), Visceral Larva Migrans and other unusual helminth infections, as well as ectoparasitic diseases such as lice (Pediculosis), Scabies, Myiasis and Tungiasis, and mites (including Chigger Syndrome; Mandell, Bennett and Dolin 2010, Principles and Practices of Infectious Diseases, 7th Edition, Elsevier Publishers, 4320 pages). There are five major species of intestinal nematodes found in humans. Because these worms spend a certain amount of time in the soil, they are sometimes known asgeohelminths. The intestinal nematodes include Ascaris lumbricoides (the large human roundworm), Enterobius vermicularis (the human threadworm or pinworm), Trichuris trichiura (human whipworm), human hookworm (Ancylostoma duodenale and Necator americanus), and Strongyloides stercoralis (threadworm).
Treatment of parasitic worms is often difficult. Ivermenctin (22, 23-dihydroavermectin B1a+22, 23-dihydroavermectin B1b), marketed under the brand name Mectizan, is currently being used to help eliminate river blindness (onchocerciasis, caused by the nematode Onchocerca volvulus) in the Americas and stop transmission of lymphatic filariasis and onchocerciasis around the world. However, the number of effective anti-parasitic therapies is few, and many would-be anti-parasitic compounds are ultimately found to be unsuitable for use in humans and other mammals or birds or wild animals, pets and livestock, because they are not effective at reaching the site of infection.
Bacteria such as Salmonella, Enterococcus, and Escherichia are known to be able to infect nematodes such as Caenorhabdus elegans, but they have not been suggested as anti-parasitic vectors capable of delivering anti-infective phage carrying peptides, antibodies, DNA or RNAs acting as a probiotic within a living host such as a human, nor has the desirability of such a system been recognized. Furthermore, the use of probiotic bacteria such has Lactococcus, Lactobacillus or Bifidobacterium have not been anticipated or considered desirable in any way for the treatment genetic modulation for the treatment of parasites. To the contrary, bacteria are thought to be used by parasitic nematodes in modulating the immune system to their own advantage (Hayes et al., 2010 Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris, Science 328: 1391-1394). New methods to deliver anti-parasitic drugs directly to the site of infection within a host would greatly enhance their effectiveness.
Viruses are among the major infectious diseases worldwide, causing massive worldwide morbidity and mortality from infections including human immunodeficiency virus (HIV), hepatitis virus, influenza virus and many others (Mandell, Bennett and Dolin 2010, Principles and Practices of Infectious Diseases, 7th Edition, Elsevier Publishers, 4320 pages). Virally infected cells may persist for extended periods of time, and new methods for treatment effective at limiting or eliminating viral infection are needed.
Cancer or neoplastic disease including solid tumor, lymphoma, leukemia or leukemic bone marrow, is a devastating condition of uncontrolled cell growth, which often has the ability to spread throughout the body (metastases) resulting in death.
Among the new modalities for a wide range of disease being explored are RNA based therapeutics, including small interfering RNA (siRNA) which results in RNA interference (RNAi) and microRNAs (miRNA). MicroRNAs (miRNA) are single-stranded RNA molecules of, for example, about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. In other instances, the therapeutic molecule is an antisense-miRNA, inhibiting the activity of an up-regulated miRNA.
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA or RNA interference (RNAi), is a class of 19-25 nucleotide-long double-stranded RNA molecules with 3′ overhangs. Asymmetric interfering RNAs have 3′ and 5′ antisense overhangs and may be only 15 base pairs in length (Sun et al. 2008 Nature Biotechnology 26: 1379-1382, incorporated in its entirety herein). Interfering RNAs play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome.
RNA interference (RNAi) is a powerful means of suppressing the expression of genes, and could potentially be used as a therapeutic intervention to suppress the expression of genes associated with disease. However, delivery of small interfering RNA (siRNA) has generally proven difficult to achieve and new delivery methodologies are eagerly sought. The present invention uses live bacterial vectors as a Trojan horse to deliver ligand modified filamentous phage carrying a siRNA cassette targeted to mammalian cells through the epidermal growth factor receptor (EGFR) and/or other receptors. These phage are packaged within gram-negative bacteria and can be carried to disease-related locations within the body such as the gut where the bacteria occur as normal flora and then be released. Furthermore, highly attenuated gram-negative bacteria such as Salmonella have the ability to target solid tumors and therefore have the potential to extend the use of RNAi for the treatment of cancer.
According to Uchida et al., 2011 in regard to the use of siRNA for therapeutic areas such the treatment of the skin (Therapeutic Effects on Atopic Dermatitis by Anti-RelA Short Interfering RNA Combined with Functional Peptides Tat and AT1002 JPET August 2011 vol. 338 no. 2 443-450) (“However, it is not known whether treatment with siRNA is an effective alternative to present medications, such as corticosteroids, and specific questions regarding the skin penetration of siRNA remain unclear. Topical application of naked siRNA does not exert strong therapeutic effects because of its low permeation efficiency owing to various skin barriers and its degradation by enzymes in the body. The most important function of the skin is to form an effective barrier between the internal and external layers of the organism.”) Thus it is apparent that delivery mechanisms that penetrate to the site of the diseased cells or tissues have the potential to overcome present limitations.
The use of live attenuated bacteria as carriers for delivering therapeutics is considered a promising methodology, yet remains without any products approved for clinical use more than 20 years after the concept was first developed (see Kotton and Hohmann 2004, Infection and Immunity 72: 5535-5547 and Roland et al., 2005, Current opinion in Molecular Therapeutics 7: 62-72 for reviews). Among the considerations for achieving therapeutic efficacy by such live attenuated bacteria delivering therapeutics is the form of the therapeutic agent, which may consist of protein, carbohydrate, DNA or RNA-based therapeutics see, e.g., U.S. Pat. Nos. 7,452,531, 7,354,592, 6,962,696, 6,923,972, 6,863,894, 6,685,935, 6,475,482, 6,447,784, 6,190,657, 6,080,849 and US Pub. 2003/0059400, each of which is expressly incorporated herein by reference. Similar hurdles also exist for therapeutic vectors secreting one or more anti-infective proteins or immunomodulatory cytokines such as IL-10 (Steidler and Rottiers, 2006, “Annals of the New York Academy of Sciences 1072:176-186.; Neirynck and Steidler 2006, Biotechnology & Genetic Engineering Reviews 22: 253-66; Steidler 2005,” Expert opinion on drug delivery 2:737-46).
RNA interference (RNAi) using small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecules is a promising technology for treatment of disease using bacterial delivery technologies (Zhang et al., 2007, Intratumoral delivery and suppression of prostate tumor growth by attenuated Salmonella enterica serovar typhimurium carrying plasmid-based small interfering RNAs Cancer Research 2007; 67: (12); Manuel et al., 2011, Enhancement of cancer vaccine therapy by systemic delivery of a tumor-targeting Salmonella-based STAT3 shRNA suppresses the growth of established melanoma tumors, Cancer Research 71(12) Jun. 15, 2011; Blache et al., 2012, Systemic delivery of Salmonella typhimurium transformed with IDS shRNA enhances intratumoral vector colonization and suppresses tumor growth, Cancer Research; 72(24) Dec. 15, 2012). Several authors have suggested the use of bacteria for delivery of RNAi (Andino-Pavlovsky et al U.S. Pat. No. 7,390,646; Andino-Pavlovsky et al. US Patent Application 2005/0118193, Xu et al., WO/2008/091375 Attenuated Salmonella as a delivery system for siRNA-based tumor therapy; Li, WO/2009/006450 Bacteria-mediated gene modulation via microRNA machinery; Li, WO/2009/006453 Enabling the use of long dsRNA for gene targeting in mammalian and other selected animal cells; Fruehauf et al., WO/2008/156702 Bacteria mediated gene silencing, Fruehauf et al., WO/2010/057009 E. coli mediated gene silencing of beta-catenin; Xu et al., WO/2008/091375 Attenuated Salmonella as a delivery system for siRNA-based tumor therapy, Raemaekers, WO/2007/083193) Methods For Controlling Pests Using RNAi, Onyabe and Hone, WO/2010/036391, A Novel RNA-Based Expression System, Gentschev et al., US Patent Application 20110287037 Microorganisms as carriers of nucleotide sequences coding for antigens and protein toxins, process of manufacture and uses thereof), each of which is expressly incorporated herein by reference in its entirety. Others have also suggested the use of siRNA for controlling parasites without using bacteria (Ward and Rhodes, WO2011/017137 Methods and compositions for treating insects and Raemaekers, WO/2007/083193) Methods For Controlling Pests Using RNAi, expressly incorporated herein by reference in its entirety).
Other regulatory RNA molecules are also recognized such as microRNA (miRNA) (Bartel, 2004, MicroRNAs: Genomics, biogenesis, mechanism and function. Cell 116: 281-297), and it has been proposed that bacteria may also have the ability to deliver miRNA (WO/2009/006450—Bacteria-Mediated Gene Modulation Via Microrna Machinery, expressly incorporated herein by reference in its entirety) and demonstrated by Yoon et al., 2010 Therapeutic effects of recombinant Salmonella typhimurium harboring CCL22 miRNA on atopic dermatitis-like skin in mice, Experimental and Molecular Medicine 43: 63-70), expressly incorporated in its entirety by reference herein.
Use of protein toxins for treatment of various disorders including inflammation, autoimmunity, neurological disorders and cancer has long-suffered from off-target toxicity. Some toxins have a natural degree of specificity for their target, such as botulinum toxin which is specific for neurons and is currently marketed as the product known as Botox® (onabotulinumtoxinA). Artificial toxin specificity has been achieved by attachment of a specific antibodies or peptide ligands (e.g., Pseudomonas endotoxin A (PE-ToxA) antibody conjugate, known as an immunotoxin). Based upon the binding specificity of the attached antibody moiety for a specific target, enhanced specificity of the target is achieved. Other toxins have been engineered to achieve specificity based upon their sight of activation. For example, aerolysin requires proteolytic activation to become cytotoxic. Substitution of the natural protease cleavage site for a tumor-specific protease cleavage site (e.g., that of the PSA protease or urokinase) results in a toxin selectively activated within tumors. However, in both these types of engineered toxins, off-target toxicity can occur. In the case of the Pseudomonas immunotoxin, several dose-limiting toxicities have been identified. Vascular leakage syndrome (VLS) is associated with hypoalbuminemia, edema, weight gain, hypotension and occasional dyspnea, which is suggested to occur by immunotoxin-mediated endothelial cell injury (Baluna et al., 2000, Exp. Cell Res. 258: 417-424), resulting in a dose-limiting toxicity. Renal injury has occurred in some patients treated with immunotoxins, which may be due to micro-aggregates of the immunotoxin (Frankel et al., 2001, Blood 98: 722a). Liver damage from immunotoxins is a frequent occurrence that is believed to be multifactorial (Frankel, 2002, Clinical Cancer Research 8: 942-944). To date, antibodies with proteinaceous toxins have limited success clinically. One explanation for the off target toxicity is that although a specific agent is targeted to the tumor and/or specifically activated there, the agent is also toxic if it diffuses out of the tumor, which is likely to occur due to the high osmotic pressure that occurs within tumors (Jain, R. K., 1994, Barriers to drug delivery in solid tumors, Scientific American 271 (11): 58-65). Once activated inside the tumor and diffused back outside, toxins such as aerolysin remain active and are able to contribute to non-target toxicity.
Protease inhibitors are known as potential drugs for treatment of diseases where proteases play a pivotal role (Turk 2006, Targeting proteases: successes, failures and future prospects. Nature Reviews Drug Discovery 5: 785-799; Motta et al., 2012; Food-grade bacteria expressing elafin protect against inflammation and restore colon homeostasis, Science Translational Medicine 4: 158 158ra144; Vergnolle et al. WO 2011/086172 Recombinant probiotic bacteria for the prevention and treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); Edwards et al., (eds) 2008, The Cancer Degradome: Proteases and Cancer Biology, Springer; Rawlings et al., 2010, MEROPS: The Peptidase Database, Nucleic Acids Res. 2010 (Database issue):D227-33, the entirety merops.sanger.ac.uk/inhibitors/which is expressly incorporated herein by reference; and Bermudes U.S. Pat. No. 8,241,623 B1 Protease sensitivity expression system, each of which is expressly incorporated herein by reference in its entirety.)
There are several protease inhibitors known in bacteria, including ecotin from E. coli (Eggers et al., 2004, The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase, Biochem J. 379: 107-118), the gene product of the inh gene in Erwinia chrysanthemi (Letoffe and Wandersman, 1989 Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi, Molecular Microbiol 3: 79-86), a protease inhibitor from Prevotella (Grenier 1994, Characteristics of a protease inhibitor produced by Prevotella intermedia FEMS Microbiol Lett 119: 13-18), Streptomyces subtilisin inhibitor (SSI) (Taguchi et al., 1990, Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene, Biochim Biophys Acta 1049: 278-285) and the Bacillus BbrPI (Shiga et al., 1991, Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene, Appl. Environ. Microbiol. 58: 525-531). However, their functions remain largely unknown (Kantyka et al., 2010, Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function, Biochimie 92: 1644-1656).